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indicate statistically significant differences. Binding of a specific protein to the CA-rich sequence Next, we used EMSA to determine whether protein(s) from Leydig cell nuclear extracts could specifically bind to the CA-rich sequence in the

placed in boiling water, which was allowed to cool at room temperature overnight. For electrophoretic mobility shift assay (EMSA), 2.5 μl COS-1 HSB extract were pre-incubated with 142 ng/μl poly(deoxyinosine-deoxycytosine) (dI.dC) for 30 min at 4 °C. For

oligonucleotides Sequences (mutated residues underlined) EMSA  WT (−209/−167) 5′-ATGCTGACGTCTTTTACTGATACCGGATCATTACGTGACTGGG-3′  Mutant −191 GATA 5′-ATGCTGACGTCTTTTACTG CC ACCGGATCATTACGTGACTGGG-3′  Mutant −205 CRE 5′-ATGCTG TG GTCTTTTACTGATACCGGATCATTACGTGACTGGG

mobility shift assay (EMSA) probes were used: PSA ARE I: 5′-GATCCTTGCAGAA CAGCAAGTGCTAGCTG-3′; 3′-GAACGTCT TGTCGTTCACGATCGACCTAG-5′; Probasin ARE II: 5′-TCGACTAGGTTCTTGGAGTACT TTG-3′; 3′-GATCCAAGAACCTCATGAAACA GCT-5′; ARE-SARG+4.6: 5′-TCGACACTGTG

II (Invitrogen), real-time PCR was performed (Bio-Rad MyiQ cycler) and relative expression levels of rOPN were determined using rL32 as internal control. Electrophoretic mobility shift assay (EMSA) EMSA was performed essentially as described ( Ausubel

electrophoretic mobility shift assays (EMSAs) to test the hypothesis that the transcription factor Sp1 mediates the stimulatory effects of insulin-mediated glucose metabolism. Finally, we incubated primary adipocyte cultures with a specific inhibitor of Sp1

-Myc promoter and identified a PTTG1 DNA-binding domain (aa 61–118) using electromobility shift assay (EMSA; Pei 2001 ). Further studies suggested that PTTG1 directly binds to the c-Myc promoter at sequences −3 and −5 and between −15 and −20 (relative to

indicated earlier. Nuclear protein was extracted from whole cells using NE-PER nuclear and cytoplasmic extraction reagents (Pierce). EMSA was performed using a LightShift Chemiluminescent EMSA kit (Pierce). In EMSA, we used previously published ( Yin et al

10 min at 4°C, the microextracts were stored at −80°C. EMSA EMSA was performed as previously described ( Giacomini et al. 2009 ). Oligonucleotides were synthesized as single strand and the annealing of the complementary strands was allowed

). In 3T3 Gene-Switch cell lines expression of HA-tagged-MN1 and HA-tag (as control) were induced overnight with mifepristone (10 − 8 M) and used to generate cell lysates for electrophoretic mobility shift assays (EMSAs). Transient transfections were